TGF-β1 is a glycoprotein belonging to a superfamily of structurally related regulatory proteins (cytokines) included within one of the three isoforms described in mammals (TGF-β 1, 2 and 3). The most abundant isoform is TGF-β1, which consists of a 25 kDa homodimer composed of two subunits joined by a disulfide bond. The amino acid sequence of human TGF-β1 has been described by authors such as Derynck K et al., “Human transforming growth factor-beta complementary DNA sequence and expression in normal and transformed cells”. Nature 316 (6030), 701-705 (1985).
TGF-β1 is a molecule with a highly preserved sequence in evolutive terms. Although it was originally defined by its capacity to induce adhesion independent of proliferation and morphological changes in rat fibroblasts, subsequent investigations have shown that TGF-β1 is a general inhibitor of proliferation of a broad range of cell types. The molecule is produced by a great variety of cell types and in different tissues during all phases of cell differentiation. It has a large series of biological effects, with the generation of potent and very often opposite effects in relation to development, physiology and immune response. Information on the role of TGF-β1 in liver regeneration and differentiation, and in liver fibrosis, as well as on the effects of the molecule upon the extracellular matrix, can be found in Spanish patent application ES 2146552 A1.
With the purpose of exploring the mechanism of action of TGF-β1, some ten proteins (membrane receptors and extracellular matrix proteins) have been reported to interact with this cytokine.
On the other hand, since many diseases or pathological alterations are associated with excessive or deregulated expression of TGF-β1, e.g., fibrosis associated to organ or tissue function loss, or surgical or esthetic complications, it is of interest to search for products capable of inhibiting the biological activity of TGF-β1—since such products can be potentially used in human or animal therapy to block the pathological consequences of excessive or deregulated TGF-β1 expression.
Several strategies have been used to inhibit the biological activity of TGF-β1 including the use of: (i) specific neutralizing antibodies; (ii) antisense oligonucleotides sequences of the gene encoding TGF-β1 which block its expression; or (iii) soluble receptors for TGF-β1 that act in a way similar to antibodies. The use of antibodies affords total and specific blockage of this cytokine (TGF-β1), though certain side effects are enhanced by both the presence of exogenous immunoglobulins in blood and the effects derived from the systemic blockage of TGF-β1. In addition, immunoglobulin stability over time does not allow short-time control of the blocking activity of this cytokine. Antisense oligonucleotides sequences inhibit TGF-β1 production at gene expression level—a fact that can generate important deregulation of all processes in which this cytokine participates.
Another strategy has recently been developed, based on the use of peptides that inhibit the biological activity of TGF-β1. In this sense, Spanish patent application ES 2146552 A1 describes some synthetic peptides originating from both TGF-β1 and its receptors, or from proteins capable of binding to TGF-β1, and which can be used as inhibitors of the biological activity of TGF-β1.
Illustrative examples of diseases or pathological alterations associated with excessive or deregulated expression of TGF-β1 include liver fibrosis, pulmonary fibrosis, corneal fibrosis and haze.
Liver fibrosis is the excessive accumulation of extracellular matrix (ECM) proteins including collagen that occurs in most types of chronic liver diseases. Advanced liver fibrosis results in cirrhosis, liver failure, and portal hypertension and often requires liver transplantation. Activated hepatic stellate cells, portal fibroblasts, and myofibroblasts of bone marrow origin have been identified as major collagen-producing cells in the injured liver. These cells are activated by fibrogenic cytokines such as TGF-β1, angiotensin II, and leptin. Reversibility of advanced liver fibrosis in patients has been recently documented, which has stimulated researchers to develop antifibrotic drugs. Emerging antifibrotic therapies are aimed at inhibiting the accumulation of fibrogenic cells and/or preventing the deposition of ECM proteins. A review summarizing recent progress in the study of the pathogenesis and diagnosis of liver fibrosis and discusses current antifibrotic strategies can be seen, for example, in Bataller R & Brenner D. A. Liver fibrosis. J Clin Invest. 2005 April; 115(4):1100.
Pulmonary fibrosis (PF) is a progressive lung disorder characterized by accumulation of ECM proteins. Unfortunately, despite its high impact on human health, no effective treatment has been yet developed. Pathogenesis of PF appears to result from a complex interaction between inflammatory cells, fibroblasts and lung parenchymal cells. Inflammatory cells produce profibrotic cytokines which cause fibroblast transformation, proliferation and accumulation of ECM proteins, causing tissue destruction and loss of lung functions. One of the most relevant profibrotic cytokines in PF is TGF-β1, which plays a key role in the synthesis and accumulation of collagen and fibronectin in lungs. This suggests that TGF-β1 inhibition would enhance the efficacy of currently used therapies. Indeed, a protective effect on the development of lung fibrosis has been described in different animal models when using anti-TGF-13 antibodies, decorin or TGF-β1 soluble receptors [Giri S N et al. Effect of antibody to transforming growth factor beta on bleomycin induced accumulation of lung collagen in mice. Thorax 1993; 48:959-966; Giri S N et al. Antifibrotic effect of decorin in a bleomycin hamster model of lung fibrosis. Biochem Pharmacol 1997; 54:1205-1216; Kolb M et al. Transient transgene expression of decorin in the lung reduces the fibrotic response to bleomycin. Am J Respir Crit Care Med 2001; 163:770-777; Wang Q et al. Reduction of bleomycin induced lung fibrosis by transforming growth factor beta soluble receptor in hamsters. Thorax 1999; 54:805-812].
Experimental evidence demonstrates that fibroblasts play a critical role in the wound-healing process and in the development of lung fibrosis. When fibroblasts become activated, they proliferate and may differentiate into myofibroblasts, displaying smooth muscle cell morphology. Different factors secreted by the pulmonary epithelium after damage, including TGF-β1, are involved in these processes inducing enhanced synthesis of ECM proteins, especially collagen and fibronectin. In fact, many in vitro studies have revealed that TGF-β1 promotes myofibroblast differentiation, induces expression of alpha-Smooth Muscle Actin (α-SMA) in lung fibroblasts and enhances the synthesis of the ECM. Interestingly, extra-domain A of fibronectin (EDA-FN), an isoform of fibronectin, is necessary for the induction of the myofibroblast phenotype by TGF-β1 in fibroblast cells. This isoform is de novo expressed during wound healing and plays an essential role in PF. In addition, areas of fibroblastic foci at sites where TGF-β1 is expressed, as well as active ECM products, have been observed in lung tissue sections from patients with idiopathic lung fibrosis.
As mentioned above, the role of TGF-β1 as one of the main fibrosis induction mediators is fully demonstrated in the scientific literature. Fibrosis in the cornea causes loss of transparency, tissue contraction and scar transformation, thus causing corneal haze. The use of both topical steroids and antimetabolites such as mitomycin C as an anti-scarring treatment is currently widespread. However, these drugs can be associated with severe complications.